Novel organic room-temperature phosphorescent probe targeting FAP (fibroblast activating protein) and application of novel organic room-temperature phosphorescent probe

Abstract

The invention discloses a novel organic room-temperature phosphorescent probe targeting fibroblast activation protein (FAP) and application thereof, and belongs to the field of molecular imaging. According to the invention, a target FAP inhibitor is coupled with a polymer shell wrapping a novel near-infrared organic room-temperature phosphorescent material TPE-4BT to obtain the organic room-temperature phosphorescent probe targeting FAP, and the organic room-temperature phosphorescent probe can be used for liver cancer imaging or preparation of reagents or drugs for liver cancer imaging. The organic room-temperature phosphorescent probe disclosed by the invention can be specifically combined with target cell fibroblasts and even can be positioned in a cell nucleus, and the observation probe in a living body can be obviously enriched in a tumor position and has no background interference problem. The organic room-temperature phosphorescent probe provided by the invention can provide accurate preoperative positioning, accurately depicts the boundary of a cancer focus during an operation, provides multi-dimensional information for accurate diagnosis and treatment of tumors, is beneficial to solving the clinical problem of liver cancer visualization, and promotes tumor transformation application.

Description

Technical Field

[0001] The invention belongs to the field of molecular imaging, and relates to a novel organic room temperature phosphorescent probe targeting fibroblast activation protein (FAP) and its construction. The invention also relates to the application of the probe in liver cancer imaging. Background Art

[0002] Liver cancer is the third leading cause of cancer-related death worldwide and ranks sixth among the most common diagnoses. More than 70% of cases are discovered in the middle and late stages. Although liver resection is the key treatment option for liver cancer, the 5-year recurrence rate can be as high as 40% to 60%. Therefore, it is of great clinical significance to develop advanced visualization technology and apply it to real-time surgical navigation.

[0003] Molecular imaging visualization technology has great potential for precise navigation in liver resection. In particular, optical imaging strategies can enhance the contrast between tumors and normal tissues using optical probes activated by tumor-specific signals. Since 2008, indocyanine green (ICG), represented by near-infrared (NIR) fluorescence imaging probes, has been applied in liver resection. Currently, the application of ICG in liver resection faces two major limitations. First, ICG fluorescence is easily quenched, resulting in a limited signal-to-noise ratio (SNR). Second, ICG fluorescence can also be observed in benign lesions, resulting in a false positive rate of 40-50%. To address these limitations, there is growing interest in the design and development of various types of probes. To date, the development of these probes is still focused on the field of fluorescence. However, due to the inherent limitations of fluorescence, such as small Stokes shift and short luminescence lifetime, it remains challenging to achieve precise and highly specific imaging. These factors, coupled with the interference of background autofluorescence, reduce the imaging quality. Organic room temperature phosphorescence (RTP) has the characteristics of large Stokes shift and long excited state lifetime due to its unique triple-state luminescence mechanism, and has received great attention as an advanced material for biological imaging. The time-resolved properties of RTP materials can effectively eliminate background interference and distinguish them from fluorescent materials, achieving highly specific and sensitive biological imaging without continuous external excitation. In order to achieve precise liver resection, it is crucial to develop RTP materials with targeted specificity.

[0004] Fibroblast activation protein (FAP) is a specific marker of cancer-associated fibroblasts (CAFs) in the tumor microenvironment, playing a role in matrix remodeling and fibrosis formation. Studies have shown that FAP is highly expressed in liver cancer and serves as an independent poor prognostic factor. Clinical studies have confirmed that targeting FAP can enhance the molecular imaging diagnosis of liver cancer and is not affected by cirrhosis. Therefore, phosphorescent probes targeting FAP are expected to become a valuable auxiliary means for navigational surgery of liver cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a novel organic room temperature phosphorescent probe targeting FAP and its preparation and application. Through the optical signal imaging provided by the specific combination of the probe and the tumor marker, it is possible to achieve accurate positioning of liver cancer, distinction of tumor boundaries, and accurate navigation of liver cancer surgery.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A novel organic room temperature phosphorescent probe targeting FAP is a nanoparticle encapsulating or loading an organic room temperature phosphorescent material and connecting a fibroblast activation protein inhibitor (FAPI) on the surface. The organic room temperature phosphorescent material is preferably TPE-4BT with the structure shown below. The preparation of TPE-4BT is described in Gu Juqing, Li Qianqian, Li Zhen, et al. Organic Materials with Ultrabright Phosphorescence at Room Temperature under Physiological Conditions for Bioimaging. Angew Chem Int Ed Engl, 2024, e202415637.

[0008]

[0009] The fibroblast activation protein inhibitor is preferably FAPI-04.

[0010] Preferably, the novel organic room temperature phosphorescent probe targeting FAP is a nanoparticle with a polymer shell encapsulating TPE-4BT and a fibroblast activation protein inhibitor (FAPI) connected to the surface. Furthermore, the hydrophilic and hydrophobic interactions between the amphiphilic polymers DSPE-PEG2000 and DSPE-PEG2000-COOH in the aqueous phase form a polymer shell encapsulating TPE-4BT; the fibroblast activation protein inhibitor can be connected to the surface of the polymer shell through reactions between amino and carboxyl groups.

[0011] The method for preparing the novel organic room temperature phosphorescent probe targeting FAP preferably comprises the following steps (the preparation schematic diagram is shown in FIG. Figure 1 A):

[0012] (1) TPE-4BT, DSPE-PEG2000 and DSPE-PEG2000-COOH are added to an organic solvent and mixed to obtain a solution 1. The solution 1 is added to water and subjected to ultrasonic treatment. After removing the organic solvent, nanoparticles TPE-4BTCOOH encapsulating TPE-4BT are obtained.

[0013] (2) The TPE-4BT COOH obtained in step (1) is reacted with NH2-FAPI-04 through a dehydration condensation reaction to obtain the novel organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP.

[0014] In step (1), the mass ratio of TPE-4BT, DSPE-PEG2000 and DSPE-PEG2000-COOH is preferably 1:5-15:0.5-1.5, more preferably 1:10:1. The organic solvent includes THF; the volume ratio of solution 1 to water is preferably 1:3-5, more preferably 1:3.

[0015] In step (2), the dehydration condensation reaction preferably uses EDC / NHS as a condensation agent.

[0016] Application of the above-mentioned novel organic room temperature phosphorescent probe targeting FAP in liver cancer imaging.

[0017] Application of the novel organic room temperature phosphorescent probe targeting FAP in the preparation of reagents or drugs for liver cancer imaging.

[0018] The present invention has the following advantages and beneficial effects:

[0019] 1. This invention is the first to couple a near-infrared novel organic room temperature phosphorescent material with a fibroblast activation protein inhibitor (FAPI), giving the material an intelligent property of targeting, significantly improving the tumor targeting capability;

[0020] 2. The probe provided by the present invention can specifically bind to the target cell fibroblasts and even localize to the cell nucleus. The probe can be significantly enriched at the tumor location when observed in vivo, which can achieve accurate localization diagnosis of subcutaneous liver cancer model. The unique time resolution property of phosphorescence can almost completely eliminate the interference of the autofluorescence background of biological samples and achieve high-quality imaging.

[0021] 3. The probe provided by the present invention can provide accurate preoperative positioning and precise delineation of cancer lesion boundaries during surgery, providing multi-dimensional information for precise diagnosis and treatment of tumors, helping to solve the clinical problem of liver cancer visualization and realize real-time navigation of in situ liver cancer resection surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The preparation flow chart and characterization results of the novel organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP. A is the preparation flow chart; B is the transmission electron microscope image and size of TPE-4BT COOH and TPE-4BT FAPI; C is the Zeta potential of TPE-4BTCOOH and TPE-4BT FAPI; D is the imaging and parameters of TPE-4BT COOH and TPE-4BT FAPI; E is the photoluminescence spectrum of TPE-4BT COOH and TPE-4BT FAPI in PBS; F is the phosphorescence spectrum of TPE-4BT COOH and TPE-4BT FAPI; G and H are the phosphorescence decay curves of TPE-4BT COOH and TPE-4BT FAPI.

[0023] Figure 2 The in vivo imaging results of the organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP in Example 2. A is the imaging flow chart of liver cancer-bearing mice; B and D are the imaging and quantitative analysis of subcutaneous liver cancer tumors in mice at different time points; C is the signal-to-noise ratio of each group; and E is the quantitative analysis of in vitro imaging of major organs and tumors.

[0024] Figure 3 This is the comparison result of in vivo imaging between the organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP in Example 3 and the clinical application probe.

[0025] Figure 4 The results of tumor resection navigation by the organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP in Example 4. A is the surgical resection process; B is the quantitative analysis of the phosphorescence intensity of the liver and tumor during the operation; and C is the pathological results of the resection and the remaining liver tissue. DETAILED DESCRIPTION

[0026] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0027] Example 1 Preparation of organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP

[0028] 1. Preparation and Characterization of Probes

[0029] (1) TPE-4BT (1 mg), DSPE-PEG2000 (10 mg) and DSPE-PEG2000-COOH (1 mg) were resuspended in THF (1 mL). The resulting solution was quickly dropped into deionized water (3 mL) and then sonicated for 1 minute using a probe sonicator equipped with a microtip (Scientz-IID). The mixture was then placed at 50°C overnight to remove the organic solvent to obtain nanoparticles TPE-4BT COOH encapsulating TPE-4BT. The aqueous solution was filtered through a PES syringe-driven filter (0.22 μm) to concentrate TPE-4BT COOH to 1 mg / mL.

[0030] (2) 0.5 mL of 1 mg / mL TPE-4BT COOH obtained in step (1), 1.303 mg N-hydroxysuccinimide (NHS), and 0.383 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were mixed and slowly stirred at room temperature for 30 min. 0.54 mg NH2-FAPI-04 was added and stirred at room temperature for 12 h. After centrifugation, the mixture was washed three times with PBS and resuspended to obtain the organic room temperature phosphorescent probe TPE-4BT FAPI.

[0031] (3) The prepared TPE-4BT COOH and TPE-4BT FAPI were characterized as follows.

[0032] Photoluminescence and excitation spectra were recorded by Hitachi F-4700 spectrofluorometer or FLS980 spectrometer. Photoluminescence lifetime and quantum yield were determined using FLS980 spectrometer. Dynamic light scattering and zeta potential measurements were performed using Malvern Nano-ZS90 Zetasizer. Transmission electron microscopy images were acquired using JEM-2100Plus transmission electron microscope. In vitro and in vivo phosphorescence imaging was performed using IVIS Spectral Imaging System (PerkinElmer Inc; charge-coupled device lens cooling temperature was −85°C).

[0033] 2. Results and Analysis

[0034] The successful connection of NH2-FAPI-04 was verified by transmission electron microscopy (TEM), and the diameter of TPE-4BT FAPI was slightly larger than that of TPE-4BT COOH. Dynamic light scattering (DLS) analysis showed that after FAPI connection, the average diameter of TPE-4BT COOH increased from 63.31nm to 66.57nm ( Figure 1 B). In addition, the zeta potentials of TPE-4BT COOH and TPE-4BT FAPI nanoparticles were -29 and -2.19 mV, respectively ( Figure 1C). The higher charge of TPE-4BT FAPI verifies the conjugation of FAPI. At the same time, the phosphorescence intensity of TPE-4BT FAPI is basically equivalent to that of TPE-4BT COOH, indicating that FAPI coupling does not affect its phosphorescence performance. There is no obvious change in the photoluminescence and phosphorescence spectra and lifetime after FAPI coupling ( Figure 1 D-1H).

[0035] Example 2 In vivo imaging of the organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP

[0036] 1. Methods

[0037] 4-week-old male nude mice weighing 20-30 g were injected subcutaneously in the right axilla with 100 μL of 5×10 6 Suspension of Huh-7 cells, a human liver malignancy cell line. Nude mice continued to be raised under specific pathogen-free conditions to maintain a stable breeding environment. Tumor formation was observed every 2 days after inoculation. After tumor formation, the tumor diameter was measured with a vernier caliper to calculate the tumor volume. Mice bearing liver cancer were injected with 1000μg / mL TPE-4BT COOH or TPE-4BT FAPI 100μL via the tail vein. Phosphorescence biological images were obtained at 4, 6, 8, 10, 24 and 48h after injection. Phosphorescence imaging was immediately acquired using a live imager after 20s of ordinary flashlight irradiation (filter on, exposure time 10 seconds).

[0038] 2. Results and Analysis

[0039] Figure 2 A shows the imaging process. No phosphorescence signal was detected in the TPE-4BT COOH group before 10 hours, which was due to its increased permeability and retention (EPR). A weak phosphorescence signal was detected in the tumor at 24 hours. Compared with the TPE-4BT COOH group, a weak signal was detected in the TPE-4BT FAPI group at 4 hours. Over time, the probe gradually accumulated in the tumor and reached a peak at the 48h time point. At the same time, at each time point, the accumulation of the probe in the tumor was more obvious in the TPE-4BT FAPI group ( Figure 2 B. Figure 2 D). The average signal-to-noise ratio of the probes coupled with the target fragment FAPI was 16 ( Figure 2 C. Figure 2 E). In vivo flashlight excitation imaging further verified the advantages of phosphorescence targeted imaging. In vitro phosphorescence imaging 48 h after injection showed that the tumor of TPE-4BT FAPI had stronger signal than that of TPE-4BT COOH. The quantitative phosphorescence intensity of the tumor of TPE-4BT FAPI was 8.53×10 5 ps -1 cm -2sr -1 .

[0040] Example 3 Comparison of imaging between the organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP and the clinical application probe

[0041] 1. Methods

[0042] The novel organic room temperature phosphorescent probe targeting FAP of the present invention was compared with the existing clinical probe ICG and ICG coupled with FAPI-04 for imaging. The structure of FAPI-ICG is as follows:

[0043]

[0044] The in vivo imaging method is described in Example 2.

[0045] 2. Results and Analysis

[0046] like Figure 3 As shown, the tumor targeting imaging capabilities of ICG and FAPI-ICG are still limited, and there is no difference in fluorescence intensity between the tumor and the background, which is not conducive to observing the location of the tumor. In addition, the coupling of ICG and FAPI will adversely affect the inherent fluorescence properties of ICG. The organic room temperature phosphorescent probe TPE-4BT FAPI targeting FAP of the present invention has obvious tumor background contrast in phosphorescence mode and has very good imaging quality.

[0047] Example 4 Organic Room Temperature Phosphorescent Probe TPE-4BT FAPI Targeting FAP to Navigate Tumor Resection

[0048] 1. Methods

[0049] 4-week-old male nude mice weighing 20-30g. After anesthesia, the nude mice were fixed on the operating table, the abdominal surgical area was disinfected, the skin was cut along the midline of the abdomen, the abdominal fascia was opened to enter the abdominal cavity, and the costal arches on both sides were gently pressed to squeeze out part of the left lobe of the liver. Then, an insulin needle was inserted along the long axis of the liver lobe at an angle of about 20° to the liver plane. After the needle was inserted, it was pushed parallel to about 1cm and 20μL of Huh-7 cell suspension was slowly injected. After injection, the needle was pulled out after 3s, and a sterile cotton swab was used to press gently until there was no active bleeding. The liver was carefully returned to the abdominal cavity, the abdomen was closed layer by layer, and the wound was disinfected. After about 2 weeks, high-frequency ultrasound can be used to evaluate whether the in situ liver cancer model was successfully constructed.

[0050] After the in situ liver cancer model is successfully constructed, the probe TPE-4BT FAPI of the present invention is used to perform phosphorescence imaging to determine the tumor resection margin. The liver cancer tissue is removed under the guidance of the tumor resection margin displayed by phosphorescence until no tumor tissue signal can be found by phosphorescence imaging. The removed tumor tissue and the remaining liver are fixed and then pathological tissue testing is performed to determine whether it is a Huh-7 tumor and a normal liver.

[0051] 2. Results and Analysis

[0052] Figure 4 A shows the surgical process. After observing the tumor position with the naked eye, phosphorescence imaging is performed. The tumor is removed under the guidance of the tumor boundary determined by phosphorescence imaging until there is no phosphorescence signal in the remaining liver. According to statistics, the intensity of phosphorescence signal in tumor is significantly higher than that in normal liver ( Figure 4 B), pathological results confirmed that the removed tissue was indeed a tumor, and the margins were negative, confirming complete removal of the tumor ( Figure 4 C).

[0053] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An organic room temperature phosphorescent probe targeting fibroblast activation protein, characterized in that: The probe is a nanoparticle that is wrapped or loaded with an organic room temperature phosphorescent material and has a fibroblast activation protein inhibitor connected to its surface; The organic room temperature phosphorescent material is TPE-4BT with the structure shown below:

2. The organic room temperature phosphorescent probe targeting fibroblast activation protein according to claim 1, characterized in that: The fibroblast activation protein inhibitor is FAPI-04.

3. The organic room temperature phosphorescent probe targeting fibroblast activation protein according to claim 1, characterized in that: The probe is a nanoparticle of TPE-4BT wrapped in a polymer shell and a fibroblast activation protein inhibitor connected on the surface.

4. The organic room temperature phosphorescent probe targeting fibroblast activation protein according to claim 3, characterized in that: The polymer shell is formed by the hydrophilic and hydrophobic interaction of DSPE-PEG2000 and DSPE-PEG2000-COOH in the water phase.

5. The organic room temperature phosphorescent probe targeting fibroblast activation protein according to claim 4, characterized in that: The fibroblast activation protein inhibitor is connected to the surface of the polymer shell through the reaction between the amino group and the carboxyl group.

6. The method for preparing the organic room temperature phosphorescent probe targeting fibroblast activation protein according to any one of claims 1 to 5, characterized in that: The following steps are included (1) TPE-4BT, DSPE-PEG2000 and DSPE-PEG2000-COOH are added to an organic solvent and mixed to obtain a solution 1. The solution 1 is added to water and subjected to ultrasonic treatment. After removing the organic solvent, nanoparticles TPE-4BTCOOH encapsulating TPE-4BT are obtained. (2) The TPE-4BT COOH obtained in step (1) is reacted with NH2-FAPI-04 through a dehydration condensation reaction to obtain the organic room temperature phosphorescent probe targeting fibroblast activation protein.

7. The method for preparing the organic room temperature phosphorescent probe targeting fibroblast activation protein according to claim 6, characterized in that: In step (1), the mass ratio of TPE-4BT, DSPE-PEG2000 and DSPE-PEG2000-COOH is 1:5-15:0.5-1.

5.

8. The method for preparing the organic room temperature phosphorescent probe targeting fibroblast activation protein according to claim 6, characterized in that: In step (1), the organic solvent includes THF; the volume ratio of solution 1 to water is 1:3-5.

9. The method for preparing the organic room temperature phosphorescent probe targeting fibroblast activation protein according to claim 6, characterized in that: In step (2), the dehydration condensation reaction uses EDC / NHS as a condensation agent.

10. Use of the organic room temperature phosphorescent probe targeting fibroblast activation protein according to any one of claims 1 to 5 in liver cancer imaging or in the preparation of an agent or drug for liver cancer imaging.

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